Fusicoccin-Like Ligands in Higher Plants

Abstract

Fusicoccin-like ligands in higher plants were sought by combining high performance liquid chromatography with radioreceptor analysis and radioimmunoassay. Such substances were found in genetically transformed cultured roots of horseradish, alteus and lupine as well as in native horseradish, cucumber, horse chestnut, and maize plants. In root crops such as carrot and sugar beet or in potato tubers, only traces of fusicoccin-like ligands were detected. Fusicoccin A was detected in genetically transformed cultured roots of horseradish by gas chromatography/mass spectrometry.The study of endogenous fusicoccins (FC) and fusicoccin-like substances has been going on for quite some while, yet the progress is rather slow. The first data on the possible presence of physiologic (Gronewald et al. 1979) and chemical (Muromtsev et al. 1980) FC analogs in higher plants were reported about 15 years after Ballio et al. (1964) found FC A in the culture medium of phytopathogenic fungus Fusicoccum amygdali Del. The finding and characterization of receptors for FC prompted a search for endogenous ligands (Aducci et al. 1980(Aducci et al. , 1995.Two distinct approaches can be used to detect endogenous bioactive substances present in minor amounts (Muromtsev 1996): (1) conventional physicochemical identification, e.g. gas chromatography/mass spectrometry (GC/MS); (2) application of highly sensitive bioaffinity tests such as radioreceptor analysis (RRA) and radioimmunoassay (RIA), to reveal specific ligands for already known receptors. In both cases the identification is carried out on samples resolved by HPLC. With RRA and RIA one can screen a broad spectrum of compounds, and their sensitivity exceeds that of GC/MS.In a series of works (Aducci et al. 1980, 1990, Ballio and Aducci 1987 endogenous ligands to FC receptors were reported in maize (Zea mays L.) roots and coleoptyles and in spinach (Spinacia oleracea) leaves. However, the amount of the isolated substance was not enough for complete chemical and biological characterization.The use of RIA for detection of hormones in higher plants was advanced by Weiler (1979), and soon after that Federico et al. (1981) applied it to FC A detection in a competitive test with [ 3 H]dihydro-FC.Since the mid 1980s we have demonstrated more than once the existence of endogenous FC A in higher plants using GC/MS analysis (Muromtsev et al. 1986, 1989. In one of the recent works (Babakov et al. 1995) we have proved the horseradish transgenic root culture to contain fusicoccin-like ligands (FCLLs), using both RRA and RIA. Our preliminary data (GC/MS) also suggested the presence of FC A in one of HPLC fractions.In this work we report the results of our further work along this line on a larger number of plants. Materials and MethodsHorseradish (Armoracia rusticana P.) plants were grown in open ground, and leaves and roots were examined. Horseradish-transformed root cultures were obtained as reported previously (Babakov et al. 1995).Seeds of cucumber (Cucumis sativus L.) and maize were germinated for 4 da...